The invention relates to modified and/or shortened forms of complement regulators derived from regulatory proteins of complement activation (RCA), especially CR1.
The complement system serves to aid in the removal of foreign substances and of immune complexes from animal hosts. This system and its regulation is reviewed by Hourcade, D., et al., Advances in Immunol (1989) 45:381-416. Briefly, the complement system generates, either by a “classical pathway” or an “alternative pathway,” C3b which binds to target immune complexes or foreign substances and marks them for destruction or clearance. C3b is generated from its precursor C3 by the proteolytic enzymes collectively designated “C3 convertase.” One form of C3 convertase is generated in the classical pathway by the association of the proteins C4b and C2a. The other form is generated in the alternative pathway by association of C3b and Bb. Both C3 convertases can associate with an additional C3b subunit to form the C5 convertases, C3bBbC3b and C4bC2aC3b, both of which are active in the production of the C5-C9 membrane attack complex which can cause cell lysis, and the production of C5a, a major proinflammatory agent.
Both C3b, and less directly, C4b, are agonists in the complement system. This is shown in the diagram in FIG. 1.
The complement system is regulated via a number of interrelated mechanisms. There are two general mechanisms for inhibition of the destructive components of the complement system. The first mechanism is generally reversible, facilitating the dissociation of the C3 convertases—i.e., C3b from Bb and C4b from C2a. Facilitation of dissociation is sometimes known as decay acceleration. The dissociation may also involve reversible binding of the antagonist proteins to C3b or C4b components, thus preventing their reassociation. The other mechanism, which is an irreversible inactivation process, results from proteolytic cleavage of the C3 convertase components C3b or C4b by the serine protease factor I. This proteolytic cleavage occurs only in the presence of a cofactor. Both general regulatory mechanisms, the facilitation of dissociation of C3b and C4b and the inactivation of C3b and C4b through cleavage by factor I, also apply to the inhibition of the alternative pathway C5 convertase (C3bBbC3b) and the classical pathway C5 convertase (C4bC2aC3b).
The proteins encoded by a region of the genome which is designated the “regulators of complement activation” (RCA) gene cluster are involved in both of the foregoing mechanisms. Currently, it is known that at least six complement proteins are encoded by this region. These are summarized in Table 1.
TABLE 1RCA Proteins: Functional ProfilPrimaryDecay AccelerationCofactorNameLigand(s)(Dissociation)ActivityCR1C3b/C4b++MCPC3b/C4b−+DAFC3b/C4b+−C3 ConvertasesC4bpC4b++Factor HC3b++CR2C3dg−ND
These proteins share certain structural similarities which are further described below.
The reversible binding to C4b or C3b to dissociate the C3 convertases is effected by two plasma proteins designated C4 binding protein (C4bp) and factor H, and by two membrane proteins designated decay acceleration factor (DAF) and complement receptor 1 (CR1). Reversible binding to C4b is effected by C4bp, DAF and CR1 while reversible binding to C3b is effected by factor H, DAF and CR1.
The irreversible inactivation of the C3 convertases resulting from proteolytic cleavage of convertase components C3b or C4b by the enzyme factor I can occur by virtue of cofactor activity effected by the above-mentioned factor H and C4bp in the plasma and by CR1 and membrane cofactor protein (MCP) at the cell surface. Cofactor activity for cleavage of C3b is effected by factor H, CR1 and MCP while cofactor activity for cleavage of C4b is effected by C4bp, CR1 and MCP. It is also possible that the sixth protein, complement receptor 2 (CR2), has this cofactor activity at the cell surface.
In summary, of the six proteins encoded by the RCA gene cluster, factor H, C4bp, and CR1 have both reversible dissociation activity and irreversible cofactor activity; DAF has only reversible dissociation activity, and MCP and possibly CR2 have only irreversible cofactor activities. CR1, DAF and MCP interact with both C3b and C4b; C4bp interacts primarily with C4b, and factor H interacts primarily with C3b.
The cDNAs corresponding to CR1, CR2, DAF, MCP, C4bp, and factor H have all been obtained and sequenced. Evaluation of these comparative sequences has lead to the alignment set forth in FIG. 2A which shows the organization of the RCA proteins into short consensus repeat (“SCR”) containing and non-SCR-containing regions with the N-terminal ends at the left. In this figure, TM refers to transmembrane domain, C to cytoplasmic domain, 0 to 0-linked glycosylation domain, G to glycolipid anchor, U to domain with unknown significance and D to a disulfide bridge-containing domain.
There is considerable uniformity among the RCA family of proteins. All of them are composed of 60-70 amino acid repeating units commonly designated “short consensus repeats” (SCRs). Each SCR shares a number of invariant or highly conserved amino acid residues with other SCRs in the same protein or SCRs in other family members. Those members of the family which are membrane bound also have at their C termini either transmembrane regions and intracellular regions or a glycolipid anchor.
The SCRs form the extracellular portions of those members of the family which are membrane-bound and almost all of the protein structure in the secreted members. Two covalently-crosslinked cysteine pairs establish two loops within each SCR. The smallest family members are DAF and MCP; each contains four SCRs followed by an 0-linked glycosylation region. DAF is terminated with a glycolipid anchor while MCP ends with an extracytoplasmic segment of unknown significance, a transmembrane region and an intracellular domain. Of the secreted members of the family, factor H contains twenty SCRs, while the native form of C4bp is an association of seven subunits of eight SCRs (the C4bp alpha chains) and one subunit of three SCRs (the C4bp beta chain). Both C4bp chains conclude with non-SCR domains that interconnect the chains through disulfide linkages. CR2 contains sixteen SCRs, a transmembrane region and an intracellular domain. The most common polymorphic form of CR1 contains four repeating units of seven similar SCRs (long homologous repeats or LHRS) numbered 1-28, followed by an additional two SCRs designated 29 and 30, a transmembrane region and an intracellular region.
Klickstein, L. B., et al., J. Exy. Med. (1988) 168:1699-1717, described the identification of distinct C3b and C4b recognition sites in CR1 using deletion mutagenesis. They concluded that a singled primary C4b binding site is located in SCR 1-2 (Sequence ID Nos. 1 and 3), while two major C3b binding sites are located in SCR 8-9 (Sequence ID Nos. 2 and 4), and SCR 15-16. C3b cofactor activity was localized to SCR 8-9 and SCR 15-16. More recently it has been shown the CR1 active site containing SCR 8-9 extends to SCR 10, and by analogy, the active site that contains SCR 15-16 (which is only one amino acid different than SCR 8-9) must extend to SCR 17. (Kalli, et al., J. Exp. Med. 174, 1451-1460 (1991); Makrides, et al., J. Biol. Chem. 267, 24754-24761 (1992)). The CR1 active site containing SCR 1-2 extends to SCR 3 and/or 4, as reported by Makrides, et al., (1992).
The murine C4bp binding site, and presumably the C4b cofactor and C4bC2a decay acceleration active sites, was reported to extend from SCRs 1-3 in the alpha chain by Ogata, et al., J. Immunology 150, 2273-2280 (1993).
The factor H binding site, and probably the C3b cofactor and C3bBb decay acceleration active sites, lies within the first five SCRs. The CR2 binding site for C3b proteolytic products extends through the first two SCRs, Kalli, et al., J. Immunology 147: 509-594 (1991); Carel, et al., J. Biol. Chem. 265: 12293-12299 (1990).
The MCP active sites extend through all four SCRs: SCRs 2-4 are required for C3b and C4b cofactor activity. SCR 1 appears unnecessary for C3b cofactor activity and binding but appears necessary for efficient C4b cofactor activity and binding, as reported by Adams, et al., J. Immunology 147:3005-3011 (1991). The DAF active sites extend through SCRs 2-4, as reported by Coyne, et al., J. Immunology 149: 2906-2913 (1992).
Hourcade, D., et al., J. Exp. Med. 168:1255-1270 (1988), described a cDNA clone designated CR1-4 that encodes the first eight and one-half amino terminal SCRs of CR1. This cDNA was transfected into COS cells which resulted in the synthesis of a secreted truncated form of CR1 with a molecular weight of 78 kd (Krych, N. et al., Proc. Natl. Acad. Sci. USA 88:4353-4357 (1991). This shortened form of the protein, as shown herein below, binds mainly C4b. This shortened form has now been determined to have C4b cofactor activity, as described herein.
The multiple binding sites of CR1 can cooperate in their interactions with C3b-containing targets. In vitro, CR1 binds C3—C3b dimers much more tightly than C3b monomers because binding to dimers can occur simultaneously at two sites in the same CR1 molecule, as reported by Wong and Farrell, J. Immunol. (1991) 146:656; Ross and Medof Adv. Immunol. (1985) 37:217). Deletion of one of the two primary C3b binding sites can reduce the binding of CR1 to C3—C3b by a factor of ten, as reported by Wong and Farrell, J. Immunol. (1991) 146:656. It is likely that the primary C4b binding site also cooperates with the primary C3b binding sites in interactions with targets that contain both C3b and C4b. These effects have an important consequence in vivo: CR1 has a higher affinity for targets densely coated with C3b and with targets densely coated with C3b plus C4b.
The C5 convertases, which are important in the stimulation of inflammation and in lysis of some target cells, are composed of multiple CR1 ligands: The classical C5 convertase contains C3b and C4b (C4bC3bC2a) while the alternative pathway C5 convertase contains two C3b proteins (C3bC3bBb). Inactivation of the C5 convertases by CR1 can also involve cooperation between more than one CR1 binding site. Wong and Farrell. J. Immunol. (1991) 146:656 showed that more than one CR1 C3b binding site may be essential for effective inhibition of alternative pathway C3 and C5 convertases.
The proteins encoded by the RCA gene cluster can be prepared recombinantly and used in diagnosis and therapy for the regulation of the complement system. The problems of transplantation of xenografts are reviewed by Platt, J. L., et al., in Immunology Today (1990) 11:450-457. Evidence has accumulated that the immediate hyperacute rejection of discordant xenografts is caused by recipient complement activity. Transgenic animals expressing human complement regulators (such as DAF or MCP) on cell surfaces could be an abundant source of organs that would be protected from hyperacute rejection in human recipients. A soluble complement inhibitor could also play a role in protecting xenografts from complement-mediated rejection.
The ability of a recombinant soluble form of CR1 to inhibit inflammation in the reversed passive Arthus reaction in rats was described by Yeh, C. G., et al., J. Immunol (1991) 146:250-256. This soluble CR1 was obtained from Chinese hamster ovary (CHO) cells expressing a CR1 genetic construct which had been mutated to remove the transmembrane and cytoplasmic domains. The ability of a similar soluble CR1, produced recombinantly in CHO cells, to inhibit post-ischemic myocardial inflammation and necrosis in rats was reported by Weissman, H. F., et al., Science (1990) 249:146-151.
Proteins related to the RCA proteins have also been shown to be produced by viruses, presumably as a mechanism whereby infection by the virus can be facilitated, as reported by Kotwaal, J., et al., Nature (1988) 335:176-178; McNearney, T. A., J Exp Med (1987) 16:1525-1535.
Complete inhibition of the complement system on a long-term basis is not likely to be desirable in most individuals. In some cases of autoimmune disease, inhibition of the classical pathway alone may be sufficient. In the case of the xenograft transplants, however, stringent inhibition of both pathways may be important. Similar stringency may be required for other applications. Accordingly, alternative modulators of the complement system with regulatable binding activities would be desirable.
It is therefore an object of the present invention to provide modified complement regulators which can be administered in soluble form for treatment of inflammatory disorders or to reduce an individuals ability to reject foreign materials.
It is a further object of the present invention to provide modified complement regulators which are shorter and more easily and economically produced than the more complex naturally occurring proteins.
It is another object of the present invention to provide complement regulators which combine the activities of different complement regulators to provide enhanced capability of inhibiting complement proteins specifically and systemically, both in the classical and alternative pathways.